backbone expressing three ms2 motifs  (Addgene inc)

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) sgRNAs harboring internal protein-binding RNA-motifs (MS2, or PP7, or Puf1) direct non-catalytic dCas9 to each targeted locus. The corresponding RNA-binding proteins (RBP) MS2, or PP7, or PUM1, are fused to mVenus, or mCherry, or iRFP670, and fluorescently label up to three different loci. For allele-specific labelling, either the 2 nd or 3 rd nucleotide in the dCas9 PAM-motif 5′-NRG-3′ was substituted by a heterozygous SNP to a non-specific dCas9-motif (IUPAC code: ‘Y’ = ‘C’ or ‘T’; ‘H’ = ‘A’, ‘C’ or ‘T’), thereby preventing dCas9-binding to either the 129S1 or CAST alleles in mouse hybrid cells. Sanger sequencing of selected SNPs confirmed heterozygosity. (b) Allele-specific visualization of 129S1- Ypel4 (yellow = MS2-mVenus) and CAST- Ypel4 (red = PP7-mCherry) in male 129S1/CAST mESCs (scale bar = 5 μm, n = 4, 35 nuclei, dashed line = mESC nucleus, arrowheads = SNP-CLING foci of maternal and paternal Ypel4 alleles). (c) Three sgRNAs in MS2 and another set of three sgRNAs in PP7 targeted the CISTR-ACT locus (sgRNA-pool 1 and 2) in RPE-1 cells to elucidate specificity. (d) All measurable foci exhibited two-color co-localization indicating locus-specificity in female RPE-1 cells (edges of foci: < 1 voxel distance = < 50 nm 3 , scale bar = 500 nm, n = 4, 35 nuclei, arrowheads = CLING foci of CISTR-ACT ). (e) To address resolution, two sgRNAs pools targeted XIST (yellow = MS2-mVenus) and TSIX (red = PP7-mCherry), separated by a topological associated domain (TAD) boundary; genomic linear distance ~69 kb. (f) Distinct, non-co-localized signals occurred in 6 out of 10 cells between XIST and TSIX , with spatial displacements of ∼163-638 nm in all three dimensions in RPE-1 cells (scale bar = 100 nm, arrowheads = CLING foci of XIST and TSIX ).

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Protein Binding, RNA Binding Assay, Binding Assay, Sequencing

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) Inter -allelic distances of Hdac4 [CI = 2.31-3.59] on largest chromosome 1 compared to a locus on small chromosome 18 (44.21 Mb, [CI = 2.14-3.19 μm]) in female 129S1/CAST MEFs were similar (each 80 nuclei, scale bars = 5 μm, arrowheads = SNP-CLING foci of maternal and paternal Hdac4 alleles). (b) Inter -allelic distances between 129S1 and CAST alleles of loci on gene dense chromosomes (chr.7:99.55 Mb [CI = 2.48-3.96 μm], chr. 11 – Sox9 [CI = 1.82-2.75 μm]) or gene-poor chromosome 15 ( Cistr-act [CI = 2.05-3.03 μm]). Chr. 11 distances were different to chr. 7 (two-tailed Mann Whitney rank sum test, * p = 0.02, each 80 nuclei, arrowheads = SNP-CLING foci of maternal and paternal Sox9 alleles). (c) Inter -allelic distances of the SNP-CLING-labeled alleles on chr.7 (arrowheads: yellow = paternal MS2-mVenus, red = maternal PP7-mCherry) relative to inter -genic distances of a CLING-labeled locus (non-allele-specific) on chr.18 (arrowheads: purple = Puf1-PUM1-iRFP670, Pearson's r 2 = 0.71, significance of Pearson's: *** p < 0.0001, 80 nuclei).

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Two Tailed Test, MANN-WHITNEY, Labeling

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) Examples of merged images demonstrate positioning of the studied alleles (arrowheads: red = PP7-mCherry, yellow = MS2-mVenus), and the nucleoli (rRNA-GFP) in 129S1/CAST MEFs (scale bars = 5 μm, n = 5). (b) Similar allelic distances and distributions of loci to the nucleoli (means ± SD), between 129S1 and CAST alleles in 129S1/CAST MEFs (normalized to nuclei sizes, each combination > 80 nuclei). (c) Examples of loci-nuclear periphery measurements (see panel a, n = 6, arrowheads = SNP-CLING foci). (d) Allelic distances and distributions of loci to the nuclear periphery were similar between 129S1 or CAST alleles (each combination > 80 nuclei). Allelic distances of the studied loci to the nucleoli (means ± SD) were significantly smaller, than their distances to the nuclear periphery (two-tailed Mann Whitney rank sum testing, * p < 0.05, *** p < 0.0001). (e) Spearman correlations of all allelic distances to the nucleoli (n = 620 alleles, r 2 = 0.329, *** p < 0.0001) or to the nuclear periphery (r 2 = 0.512, *** p < 0.0001). 61 % of 129S1 or 64 % CAST inherited loci were closer than 1 μm to the nucleolus. 35 % of 129S1 or 30 % of CAST loci were closer than 1 μm to the periphery. (f) Number of nucleoli counted in human RPE-1 or MEFs was higher in MEFs (two-tailed Mann Whitney rank sum testing, *** p < 0.0001). (g) Similar inter -nucleoli distances in human RPE-1 or MEFs (Kruskal-Wallis multiple comparisons testing [not adjusted], ns = not significant, medians with 25 th to 75 th percentiles, min/max, and sample sizes [n] are depicted, RPE-1 CI lower = 3.02-3.31 μm, CI upper = 3.67-3.99 μm, MEFs CI lower = 3.06-3.35 μm, CI upper = 3.63-4.7 μm).

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) Example of CLING-labeled FIRRE loci (arrowheads: red = PP7-mCherry, 108 nuclei) and their distances to the closest nucleolus (rRNA-GFP) in female RPE-1 cells (scale bars = 5 μm). Loci-nucleoli distances revealed that FIRRE was closer to the perinucleolar space than GAPDH (107 nuclei, two-tailed Mann-Whitney rank sum test, *** p < 0.0001). (b) Example of maternal and paternal Firre alleles (arrowheads) in female 129S1 (yellow = MS2-mVenus), CAST (red = PP7-mCherry) MEFs. Firre's allelic distances to the nucleoli (129S1 CI = 0.77-1.32 μm, CAST CI = 0.81-1.38 μm) or to the nuclear periphery (80 nuclei, means ± SD, 129S1 CI = 1.38-1.84 μm, CAST CI = 1.48-1.89 μm). Firre was closer to the nucleoli than to the nuclear periphery (two-tailed Mann Whitney rank sum test, *** p < 0.0001). (c) Scheme of interacting loci between two non-homologous chromosomes in spatial proximity, targeted by SNP-CLING or CLING. (d) SNP-CLING of maternal 129S1-derived Firre (yellow = MS2-mVenus) with either Ypel4 -129S1 or Ypel4 -CAST (red = PP7-mCherry) in male mESCs (n = 8, arrowheads = SNP-CLING foci). (e) Allele-biased interaction of Firre with the paternal Ypel4 -CAST locus (** p = 0.0012, Χ 2 -test, each quantification > 130 nuclei, n = 5) in male 129S1/CAST mESCs.

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Labeling, Two Tailed Test, MANN-WHITNEY, Derivative Assay